-The main virulence factor of Streptococcus pneumoniae is the capsular polysaccharide (PS), which is the antigen of all current vaccines that are prepared with PS purified from serotypes prevalent in the population. In this work, three purification strategies were evaluated and a new process was developed for purification of serotype 14 PS (PS14), responsible for 39.8% of diseases in children of 0-6 years old in Brazil. The developed method consists of cell separation by tangential microfiltration, concentration of the microfiltrate by tangential ultrafiltration (50 kDa), diafiltration in the presence of sodium dodecyl sulfate using a 30 kDa ultrafiltration membrane, precipitation with 5% trichloroacetic acid, precipitation with 20% and 60% ethanol, and anion exchange chromatography. The required purity regarding nucleic acids (≤ 2%) and proteins (≤ 3%) was achieved, resulting in a relative purity of 439 mg PS14/mg nucleic acids and 146 mg PS14/mg proteins. The final polysaccharide recovery was 65%, which is higher than the recovery of the majority of processes described in the literature.
Streptococcus pneumoniae is a human pathogen largely transmitted by aerosols. Vaccines are the main strategy against this pathogen, and the capsular polysaccharide (PS) is its major antigen. S. pneumoniae serotype 1 is associated with large outbreaks and epidemics of invasive diseases. The aims of this work were to screen serotype 1 strains to identify the best PS1 producer, evaluate three peptones for PS1 production, investigate the effects of culture medium components using a design of experiments (DoE), a statistic tool for optimization, and propose a new medium/cultivation strategy. After flask cultivation of nine strains, two that produced high PS1 and biomass values were chosen for further evaluation in the bioreactor, and ST595/01 was chosen as the best PS1 producer strain. Among the peptones tested (Casamino acids, Soytone, and Phytone), the highest PS1 production (298 mg/L) was reached with Phytone. Next, DoE (2(4-1)) was performed to evaluate the effects of yeast extract (YE), Phytone, L-asparagine (Asn), and L-glutamine (Gln), yielding the following results: Phytone presented positive effects (p < 0.05) for maximum production of biomass, PS1, acetate, and lactate; YE showed positive effects for biomass and acid production (p < 0.05); Gln exerted a minor positive effect on PS1 yield factor on glucose (p < 0.1); and Asn presented only an effect on acetate production (p < 0.1). Hence, a new culture medium was formulated based on Phytone, YE, and glucose, and batch and fed-batch cultivations were evaluated. The fed-batch cultivation showed almost 2 times the biomass and 2.5 times the PS1 production as the batch culture, and 8-10 times higher PS1 production than has been previously reported.
Título da Dissertação: Desenvolvimento de meio quimicamente definido para produção de polissacarídeo capsular em cultivo de Streptococcus pneumoniae sorotipo 14.
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